Human coagulation factor VII polypeptides

ABSTRACT

The present invention relates to novel human coagulation Factor VIIa variants having coagulant activity as well as polynucleotide constructs encoding such variants, vectors and host cells comprising and expressing the polynucleotide, pharmaceutical compositions, uses and methods of treatment.

FIELD OF THE INVENTION

[0001] The present invention relates to novel human coagulation FactorVIIa polypeptides having coagulant activity as well as polynucleotideconstructs encoding such polypeptides, vectors and host cells comprisingand expressing the polynucleotide, pharmaceutical compositions, uses andmethods of treatment.

BACKGROUND OF THE INVENTION

[0002] Blood coagulation is a process consisting of a complexinteraction of various blood components (or factors) that eventuallygives raise to a fibrin clot. Generally, the blood components, whichparticipate in what has been referred to as the coagulation “cascade”,are enzymatically inactive proteins (proenzymes or zymogens) that areconverted to proteolytic enzymes by the action of an activator (whichitself is an activated clotting factor). Coagulation factors that haveundergone such a conversion are generally referred to as “activefactors”, and are designated by the addition of the letter “a” to thename of the coagulation factor (e.g. Factor VIIa).

[0003] Initiation of the haemostatic process is mediated by theformation of a complex between tissue factor, exposed as a result ofinjury to the vessel wall, and Factor VIIa. This complex then convertsFactors IX and X to their active forms. Factor Xa converts limitedamounts of prothrombin to thrombin on the tissue factor-bearing cell.Thrombin activates platelets and Factors V and VIII into Factors Va andVIIIa, both cofactors in the further process leading to the fullthrombin burst. This process includes generation of Factor Xa by FactorIXa (in complex with factor VIIIa) and occurs on the surface ofactivated platelets. Thrombin finally converts fibrinogen to fibrinresulting in formation of a fibrin clot. In recent years Factor VII andtissue factor have been found to be the main initiators of bloodcoagulation.

[0004] Factor VII is a trace plasma glycoprotein that circulates inblood as a single-chain zymogen. The zymogen is catalytically inactive.Single-chain Factor VII may be converted to two-chain Factor VIIa byFactor Xa, Factor XIIa, Factor IXa, Factor VIIa or thrombin in vitro.Factor Xa is believed to be the major physiological activator of FactorVII. Like several other plasma proteins involved in haemostasis, FactorVII is dependent on Vitamin K for its activity, which is required forthe gamma-carboxylation of multiple glutamic acid residues that areclustered close to the amino terminus of the protein. Thesegamma-carboxylated glutamic acids are required for the metal ion-inducedinteraction of Factor VII with phospholipids. The conversion of zymogenFactor VII into the activated two-chain molecule occurs by cleavage ofan internal Arg₁₅₂-Ile₁₅₃ peptide bond. In the presence of tissuefactor, phospholipids and calcium ions, the two-chain Factor VIIarapidly activates Factor X or Factor IX by limited proteolysis.

[0005] It is often desirable to stimulate or improve the coagulationcascade in a subject. Factor VIIa has been used to control bleedingdisorders that have several causes such as clotting factor deficiencies(e.g. haemophilia A and B or deficiency of coagulation Factors XI orVII) or clotting factor inhibitors. Factor VIIa has also been used tocontrol excessive bleeding occurring in subjects with a normallyfunctioning blood clotting cascade (no clotting factor deficiencies orinhibitors against any of the coagulation factors). Such bleeding may,for example, be caused by a defective platelet function,thrombocytopenia or von Willebrand's disease. Bleeding is also a majorproblem in connection with surgery and other forms of tissue damage.

[0006] European Patent No. 200,421 (ZymoGenetics) relates to thenucleotide sequence encoding human Factor VII and the recombinantexpression of Factor VII in mammalian cells.

[0007] Dickinson et al. (Proc. Natl. Acad. Sci. USA (1996) 93,14379-14384) relates to a Factor VII variant wherein Leu305 has beenreplaced by Ala (FVII(Ala305)).

[0008] Iwanaga et al. (Thromb. Haemost. (supplement August 1999), 466,abstract 1474) relates to Factor VIIa variants wherein residues 316-320are deleted or residues 311-322 are replaced with the correspondingresidues from trypsin.

[0009] There is a need for variants of Factor VIIa having coagulantactivity, variants with high activity that can be administered atrelatively low doses, and variants which do not produce the undesirableside effects such as systemic activation of the coagulation system andbleeding, respectively, associated with conventional therapies.

DESCRIPTION OF THE INVENTION

[0010] It has now been found that human coagulation Factor VIIapolypeptide variants wherein the amino acid Leu305 and at least oneamino acid independently selected from the group consisting of Lys157,Lys337, Asp334, Ser336, Val158, Glu296, and Met298 of SEQ ID NO:1 arereplaced by different amino acids have increased coagulant activitycompared to wild type human coagulation Factor VIIa.

[0011] The term “a different amino acid” as used herein means one aminoacid that are different from that amino acid naturally present at thatposition. This includes but are not limited to amino acids that can beencoded by a polynucleotide. Preferably the different amino acid is innatural L-form and can be encoded by a polynucleotide. A specificexample being L-cysteine (Cys).

[0012] The term “activity” as used herein means the ability of a FactorVII polypeptide to convert its substrate Factor X to the active FactorXa. The activity of a Factor VII polypeptide may be measured with the“In Vitro Proteolysis Assay” (see Example 6).

[0013] The term “inherent activity” also includes the ability togenerate thrombin on the surface of activated platelets in the absenceof tissue factor.

[0014] The Leu305 is located at the end of an α-helix found in thetissue factor-complexed form of Factor VIIa, which is believed to beimportant to the activity. In free Factor VIIa (Factor VIIa not bound totissue factor) the helix is distorted and thus possibly unstable. Thepolypeptide variants according to the present invention attain theactive conformation, which normally has to be induced by tissue factor.The increased activity of the polypeptide variants compared to wild typeFactor VIIa may be due to a stabilisation of the α-helix, areorientation of the helix or some other change in conformation.Replacement of the Leu305 will induce a reorientation and/orstabilisation of the helix.

[0015] The amino acids comprising Lys157, Lys337, Asp334, Ser336,Val158, Glu296, and Met298 are located in an area believed to affect theinsertion of the amino terminus of the protease domain and thereby theformation of the catalytically active conformation of Factor VIIa whichis dependent on a salt bridge between the terminal amino group of Ile153and the side chain of Asp343. The replacements may remove electrostaticrepulsions, add hydrogen bonds or otherwise facilitate the insertion ofthe amino terminus.

[0016] Due to the higher inherent activity of the described Factor VIIapolypeptide variants compared to native FVIIa, a lower dose may beadequate to obtain a functionally adequate concentration at the site ofaction and thus it will be possible to administer a lower dose to thesubject having bleeding episodes or needing enhancement of the normalhaemostatic system.

[0017] It has been found by the present inventors that by replacing theamino acid Leu305 in combination with one or more of the Lys in position157 and the Lys in position 337 and the Val in position 158 and the Gluin position 296 and the Met in position 298 and the Asp in position 334and the Ser in position 336, Factor VIIa will spontaneously attain amore active conformation that normally has to be induced by tissuefactor. Such Factor VIIa polypeptide variants exhibit an inherentactivity which may be therapeutically useful in situations where theprocoagulant activity is independent of tissue factor (Factor Xageneration on the platelet surface) such as when high doses of, forexample, NovoSeven® are administered.

[0018] In a further embodiment additional replacement of amino acids inthe protease domain further facilitate formation of the activeconformation of the molecule. It is believed, however, that the mostpronounced effects will be seen when the above-mentioned mutations arecarried out in the vicinity (sequential or three-dimensional) of theselatter seven amino acids.

[0019] The invention further comprises replacement of a few amino acidsin the N-terminal Gla domain (amino acids at position corresponding to1-37 of SEQ ID NO:1) of Factor VIIa can provide the protein with asubstantially higher affinity for membrane phospholipids, such asmembrane phospholipids of tissue factor-bearing cells or of platelets,thereby generating Factor VII polypeptide variants which have animproved procoagulant effect.

[0020] Thus, the Factor VIIa polypeptide variants mentioned above may,in addition to the already performed amino acid replacement in position305 in combination with replacements in positions 157, 158, 296, 298,334, 336 or 337 and the optional amino acid replacements elsewhere inthe protease domain, also have at least one amino acid replaced in theN-terminal Gla domain, thereby obtaining a protein having an increasedactivity as well as an increased affinity for membrane phospholipidscompared to native Factor VII. Preferably the amino acids in positions10 and 32 (referring to SEQ ID NO:1) of Factor VII may be replaced witha different amino acid. Examples of preferred amino acids to beincorporated in the above-mentioned positions are: The amino acid Pro inposition 10 is replaced by Gln, Arg, His, Gln, Asn or Lys; and/or theamino acid Lys in position 32 is replaced by Glu, Gln or Asn.

[0021] Other amino acids in the Gla domain, based on the differentphospholipid affinities and sequences of the vitamin K-dependent plasmaproteins, may also be considered for substitution.

[0022] The term “N-terminal GLA-domain” means the amino acid sequence1-37 of Factor VII.

[0023] The three-letter indication “GLA” means 4-carboxyglutamic acid(γ-carboxyglutamate).

[0024] The term “protease domain” means the amino acid sequence 153-406of Factor VII (the heavy-chain of Factor VIIa).

[0025] The term “Factor VII polypeptide” as used herein means anyprotein comprising the amino acid sequence 1-406 of native human FactorVII (SEQ ID NO: 1) or variants thereof. This includes but are notlimited to human Factor VII, human Factor VIIa and variants thereof.

[0026] The term “Factor VII” as used herein is intended to comprise theinactive one-chain zymogen Factor VII molecule as well as the activatedtwo-chain Factor VII molecule (Factor VIIa). This includes proteins thathave the amino acid sequence 1-406 of native human Factor VII or FactorVIIa. It also includes proteins with a slightly modified amino acidsequence, for instance, a modified N-terminal end including N-terminalamino acid deletions or additions so long as those proteinssubstantially retain the activity of Factor VIIa. The term “factorVIIa”, or “FVIIa” as used herein means a product consisting of theactivated form (factor VIIa). “Factor VII” or “Factor VIIa” within theabove definition also includes natural allelic variations that may existand occur from one individual to another. Also, degree and location ofglycosylation or other post-translation modifications may vary dependingon the chosen host cells and the nature of the host cellularenvironment.

[0027] The terms “variant” or “variants”, as used herein, is intended todesignate Factor VII having the sequence of SEQ ID NO:1, wherein one ormore amino acids of the parent protein have been substituted by anotheramino acid and/or wherein one or more amino acids of the parent proteinhave been deleted and/or wherein one or more amino acids have beeninserted in protein and/or wherein one or more amino acids have beenadded to the parent protein. Such addition can take place either at theN-terminal end or at the C-terminal end of the parent protein or both.The “variant” or “variants” within this definition still have FVIIactivity in its activated form. In one embodiment a variant is 70%identical with the sequence of of SEQ ID NO:1. In one embodiment avariant is 80% identical with the sequence of of SEQ ID NO:1. In anotherembodiment a variant is 90% identical with the sequence of of SEQ IDNO:1. In a further embodiment a variant is 95% identical with thesequence of of SEQ ID NO: 1.

[0028] In a first aspect, the invention relates to a Factor VIIpolypeptide comprising at least two substitutions relative to the aminoacid sequence of SEQ ID NO:1, wherein said substitutions are (i)replacement of L305 with any other amino acid, and (ii) replacement withany other amino acid of one or more amino acids selected from the groupconsisting of K157, K337, D334, S336, V158, E296, and M298.

[0029] In a second aspect, the invention relates to a Factor VIIpolypeptide with two substitutions relative to the amino acid sequenceof SEQ ID NO:1, wherein said substitutions are (i) replacement of L305with any other amino acid and (ii) replacement with any other amino acidof one amino acid selected from the group consisting of K157, K337,D334, S336, V158, E296, and M298.

[0030] In a third aspect, the invention relates to a Factor VIIpolypeptide with three substitutions relative to the amino acid sequenceof SEQ ID NO:1, wherein said substitutions are (i) replacement of L305with any other amino acid and (ii) replacement with any other amino acidof two amino acids selected from the group consisting of K157, K337,D334, S336, V158, E296, and M298.

[0031] In a further aspect, the invention relates to a Factor VIIpolypeptide with four substitutions relative to the amino acid sequenceof SEQ ID NO:1, wherein said substitutions are (i) replacement of L305with any other amino acid and (ii) replacement with any other amino acidof three amino acids selected from the group consisting of K157, K337,D334, S336, V158, E296, and M298.

[0032] In a further aspect, the invention relates to a Factor VIIpolypeptide with five substitutions relative to the amino acid sequenceof SEQ ID NO:1, wherein said substitutions are (i) replacement of L305with any other amino acid and (ii) replacement with any other amino acidof four amino acids selected from the group consisting of K157, K337,D334, S336, V158, E296, and M298.

[0033] In a further aspect, the invention relates to a Factor VIIpolypeptide with six substitutions relative to the amino acid sequenceof SEQ ID NO:1, wherein said substitutions are (i) replacement of L305with any other amino acid and (ii) replacement with any other amino acidof five amino acids selected from the group consisting of K157, K337,D334, S336, V158, E296, and M298.

[0034] In a further aspect, the invention relates to a Factor VIIpolypeptide with seven substitutions relative to the amino acid sequenceof SEQ ID NO:1, wherein said substitutions are (i) replacement of L305with any other amino acid and (ii) replacement with any other amino acidof six amino acids selected from the group consisting of K157, K337,D334, S336, V158, E296, and M298.

[0035] In a further aspect, the invention relates to a Factor VIIpolypeptide with eight substitutions relative to the amino acid sequenceof SEQ ID NO:1, wherein said substitutions are (i) replacement of L305with any other amino acid and (ii) replacement with any other amino acidof the amino acids K157, K337, D334, S336, V158, E296, and M298.

[0036] In a further aspect, the invention relates to a polynucleotideconstruct encoding a Factor VII polypeptide comprising at least twosubstitutions relative to the amino acid sequence of SEQ ID NO:1,wherein said substitutions are (i) replacement of L305 with any otheramino acid, and (ii) replacement with any other amino acid of one ormore amino acids selected from the group consisting of K157, K337, D334,S336, V158, E296, and M298.

[0037] The term “construct” is intended to indicate a polynucleotidesegment which may be based on a complete or partial naturally occurringnucleotide sequence encoding the polypeptide of interest. The constructmay optionally contain other polynucleotide segments. In a similar way,the term “amino acids which can be encoded by polynucleotide constructs”covers amino acids which can be encoded by the polynucleotide constructsdefined above, i.e. amino acids such as Ala, Val, Leu, Ile, Met, Phe,Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.

[0038] In a further aspect, the invention provides a recombinant vectorcomprising the polynucleotide construct encoding a Factor VIIpolypeptide.

[0039] The term “vector”, as used herein, means any nucleic acid entitycapable of the amplification in a host cell. Thus, the vector may be anautonomously replicating vector, i.e. a vector, which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated. The choice of vector will often depend onthe host cell into which it is to be introduced. Vectors include, butare not limited to plasmid vectors, phage vectors, viruses or cosmidvectors. Vectors usually contains a replication origin and at least oneselectable gene, i.e., a gene which encodes a product which is readilydetectable or the presence of which is essential for cell growth.

[0040] In a further aspect, the invention provides a recombinant hostcell comprising the polynucleotide construct or the vector. In oneembodiment the recombinant host cell is a eukaryotic cell. In anotherembodiment the recombinant host cell is of mammalian origin. In afurther embodiment the recombinant host cell is selected from the groupconsisting of CHO cells, HEK cells and BHK cells.

[0041] The term “a host cell”, as used herein, represent any cell,including hybrid cells, in which heterologous DNA can be expressed.Typical host cells includes, but are not limited to insect cells, yeastcells, mammalian cells, including human cells, such as BHK, CHO, HEK,and COS cells. In practicing the present invention, the host cells beingcultivated are preferably mammalian cells, more preferably anestablished mammalian cell line, including, without limitation, CHO(e.g., ATCC CCL 61), COS-1 (e.g., ATCC CRL 1650), baby hamster kidney(BHK) and HEK293 (e.g., ATCC CRL 1573; Graham et al., J. Gen. Virol.36:59-72, 1977) cell lines. A preferred BHK cell line is the tk⁻ ts13BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA79:1106-1110, 1982), hereinafter referred to as BHK 570 cells. The BHK570 cell line is available from the American Type Culture Collection,12301 Parklawn Dr., Rockville, Md. 20852, under ATCC accession numberCRL 10314. A tk⁻ ts13 BHK cell line is also available from the ATCCunder accession number CRL 1632. Other suitable cell lines include,without limitation, Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II(Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB8065), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (Urlaub and Chasin, Proc.Natl. Acad. Sci. USA 77:4216-4220, 1980). Also useful are 3T3 cells,Namalwa cells, myelomas and fusions of myelomas with other cells.

[0042] In a further aspect, the invention provides a transgenic animalcontaining and expressing the polynucleotide construct.

[0043] In a further aspect, the invention provides a transgenic plantcontaining and expressing the polynucleotide construct.

[0044] In a further aspect, the invention relates to a method forproducing the Factor VII polypeptide of the invention, the methodcomprising cultivating a cell comprising the polynucleotide construct inan appropriate growth medium under conditions allowing expression of thepolynucleotide construct and recovering the resulting polypeptide fromthe culture medium.

[0045] As used herein the term “appropriate growth medium” means amedium containing nutrients and other components required for the growthof cells and the expression of the nucleic acid sequence encoding theFactor VII polypeptide of the invention.

[0046] In a further aspect, the invention relates to a method forproducing the Factor VII polypeptide, the method comprising recoveringthe polypeptide from milk produced by the transgenic animal.

[0047] In a further aspect, the invention relates to a method forproducing the Factor VII polypeptide, the method comprising cultivatinga cell of a transgenic plant comprising the polynucleotide construct,and recovering the polypeptide from the resulting plant.

[0048] In a further aspect, the invention relates to a pharmaceuticalcomposition comprising a Factor VII polypeptide comprising at least twosubstitutions relative to the amino acid sequence of SEQ ID NO:1,wherein said substitutions are (i) replacement of L305 with any otheramino acid, and (ii) replacement with any other amino acid of one ormore amino acids selected from the group consisting of K157, K337, D334,S336, V158, E296, and M298; and, optionally, a pharmaceuticallyacceptable carrier.

[0049] In a further aspect, the invention relates to the use of a FactorVII polypeptide comprising at least two substitutions relative to theamino acid sequence of SEQ ID NO:1, wherein said substitutions are (i)replacement of L305 with any other amino acid, and (ii) replacement withany other amino acid of one or more amino acids selected from the groupconsisting of K157, K337, D334, S336, V158, E296, and M298; for thepreparation of a medicament. In one embodiment the medicament is for thetreatment of bleeding disorders or bleeding episodes or for theenhancement of the normal haemostatic system. In one embodiment the useis for the treatment of haemophilia A or B.

[0050] In the present context, the term “treatment” is meant to includeboth prevention of an expected bleeding, such as in surgery, andregulation of an already occurring bleeding, such as in trauma, with thepurpose of inhibiting or minimising the bleeding. Prophylacticadministration of the Factor VIIa polypeptide according to the inventionis thus included in the term “treatment”.

[0051] The term “bleeding episodes” is meant to include uncontrolled andexcessive bleeding. Bleeding episodes may be a major problem both inconnection with surgery and other forms of tissue damage. Uncontrolledand excessive bleeding may occur in subjects having a normal coagulationsystem and subjects having coagulation or bleeding disorders. As usedherein the term “bleeding disorder” reflects any defect, congenital,acquired or induced, of cellular or molecular origin that is manifestedin bleedings. Examples are clotting factor deficiencies (e.g.haemophilia A and B or deficiency of coagulation Factors XI or VII),clotting factor inhibitors, defective platelet function,thrombocytopenia or von Willebrand's disease.

[0052] Excessive bleedings also occur in subjects with a normallyfunctioning blood clotting cascade (no clotting factor deficiencies or-inhibitors against any of the coagulation factors) and may be caused bya defective platelet function, thrombocytopenia or von Willebrand'sdisease. In such cases, the bleedings may be likened to those bleedingscaused by haemophilia because the haemostatic system, as in haemophilia,lacks or has abnormal essential clotting “compounds” (such as plateletsor von Willebrand factor protein) that causes major bleedings. Insubjects who experience extensive tissue damage in association withsurgery or vast trauma, the normal haemostatic mechanism may beoverwhelmed by the demand of immediate haemostasis and they may developbleeding in spite of a normal haemostatic mechanism. Achievingsatisfactory haemostasis also is a problem when bleedings occur inorgans such as the brain, inner ear region and eyes with limitedpossibility for surgical haemostasis. The same problem may arise in theprocess of taking biopsies from various organs (liver, lung, tumourtissue, gastrointestinal tract) as well as in laparoscopic surgery.Common for all these situations is the difficulty to provide haemostasisby surgical techniques (sutures, clips, etc.) which also is the casewhen bleeding is diffuse (haemorrhagic gastritis and profuse uterinebleeding). Acute and profuse bleedings may also occur in subjects onanticoagulant therapy in whom a defective haemostasis has been inducedby the therapy given. Such subjects may need surgical interventions incase the anticoagulant effect has to be counteracted rapidly. Radicalretropubic prostatectomy is a commonly performed procedure for subjectswith localized prostate cancer. The operation is frequently complicatedby significant and sometimes massive blood loss. The considerable bloodloss during prostatectomy is mainly related to the complicatedanatomical situation, with various densely vascularized sites that arenot easily accessible for surgical haemostasis, and which may result indiffuse bleeding from a large area. Another situation that may causeproblems in the case of unsatisfactory haemostasis is when subjects witha normal haemostatic mechanism are given anticoagulant therapy toprevent thromboembolic disease. Such therapy may include heparin, otherforms of proteoglycans, warfarin or other forms of vitamin K-antagonistsas well as aspirin and other platelet aggregation inhibitors.

[0053] In one embodiment of the invention, the bleeding is associatedwith haemophilia. In another embodiment, the bleeding is associated withhaemophilia with aquired inhibitors. In another embodiment, the bleedingis associated with thrombocytopenia. In another embodiment, the bleedingis associated with von Willebrand's disease. In another embodiment, thebleeding is associated with severe tissue damage. In another embodiment,the bleeding is associated with severe trauma. In another embodiment,the bleeding is associated with surgery. In another embodiment, thebleeding is associated with laparoscopic surgery. In another embodiment,the bleeding is associated with haemorrhagic gastritis. In anotherembodiment, the bleeding is profuse uterine bleeding. In anotherembodiment, the bleeding is occurring in organs with a limitedpossibility for mechanical haemostasis. In another embodiment, thebleeding is occurring in the brain, inner ear region or eyes. In anotherembodiment, the bleeding is associated with the process of takingbiopsies. In another embodiment, the bleeding is associated withanticoagulant therapy.

[0054] The term “subject” as used herein is intended to mean any animal,in particular mammals, such as humans, and may, where appropriate, beused interchangeably with the term “patient”.

[0055] The term “enhancement of the normal haemostatic system” means anenhancement of the ability to generate thrombin.

[0056] In a further aspect, the invention relates to a method for thetreatment of bleeding disorders or bleeding episodes in a subject or forthe enhancement of the normal haemostatic system, the method comprisingadministering a therapeutically or prophylactically effective amount ofa Factor VII polypeptide comprising at least two substitutions relativeto the amino acid sequence of SEQ ID NO:1, wherein said substitutionsare (i) replacement of L305 with any other amino acid, and (ii)replacement with any other amino acid of one or more amino acidsselected from the group consisting of K157, K337, D334, S336, V158,E296, and M298; to a subject in need thereof.

[0057] In a further aspect, the invention relates to the Factor VIIpolypeptide of the invention for use as a medicament.

[0058] In one embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein L305 is replaced with any other amino acid andK157 is replaced with any other amino acid.

[0059] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein L305 is replaced with any otheramino acid and K337 is replaced with any other amino acid.

[0060] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein L305 is replaced with any otheramino acid and D334 is replaced with any other amino acid.

[0061] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein L305 is replaced with any otheramino acid and S336 is replaced with any other amino acid.

[0062] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein L305 is replaced with any otheramino acid and V158 is replaced with any other amino acid.

[0063] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein L305 is replaced with any otheramino acid and E296 is replaced with any other amino acid.

[0064] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein L305 is replaced with any otheramino acid and M298 is replaced with any other amino acid.

[0065] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein at least one amino acid in theremaining positions in the protease domain has been replaced with anyother amino acid.

[0066] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein at least one amino acid in theremaining positions in the protease domain has been replaced with anyother amino acid.

[0067] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein at the most 20 additional aminoacids in the remaining positions in the protease domain have beenreplaced with any other amino acids.

[0068] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein at least one amino acidcorresponding to an amino acid at a position selected from 159-170 ofSEQ ID NO:1 has been replaced with any other amino acid.

[0069] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein at least one amino acidcorresponding to an amino acid at a position selected from 290-304 ofSEQ ID NO:1 has been replaced with any other amino acid.

[0070] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein R304 has been replaced by an aminoacid selected from the group consisting of Tyr, Phe, Leu, and Met.

[0071] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein at least one amino acidcorresponding to an amino acid at a position selected from 306-312 ofSEQ ID NO:1 has been replaced with any other amino acid.

[0072] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein M306 has been replaced by an aminoacid selected from the group consisting of Asp, and Asn.

[0073] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein D309 has been replaced by an aminoacid selected from the group consisting of Ser, and Thr.

[0074] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein at least one amino acidcorresponding to an amino acid at a position selected from 330-339 ofSEQ ID NO:1 has been replaced with any other amino acid.

[0075] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein A274 has been replaced with anyother amino acid.

[0076] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein the A274 has been replaced by anamino acid selected from the group consisting of Met, Leu, Lys, and Arg.

[0077] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein the K157 has been replaced by anamino acid selected from the group consisting of Gly, Val, Ser, Thr,Asn, Gln, Asp, and Glu.

[0078] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein said K337 has been replaced by anamino acid selected from the group consisting of Ala, Gly, Val, Ser,Thr, Asn, Gln, Asp, and Glu.

[0079] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein said D334 has been replaced by anamino acid selected from the group consisting of Gly, and Glu.

[0080] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein said S336 has been replaced by anamino acid selected from the group consisting of Gly, and Glu.

[0081] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein said V158 has been replaced by anamino acid selected from the group consisting of Ser, Thr, Asn, Gln,Asp, and Glu.

[0082] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein said E296 has been replaced by anamino acid selected from the group consisting of Arg, Lys, and Val.

[0083] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein said M298 has been replaced by anamino acid selected from the group consisting of Lys, Arg, Gln, and Asn.

[0084] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein said L305 has been replaced by anamino acid selected from the group consisting of Val, Tyr and Ile.

[0085] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein said L305 has been replaced byVal.

[0086] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein the amino acid has been replacedby a different amino acid which can be encoded by polynucleotideconstructs.

[0087] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein said Factor VII polypeptide ishuman Factor VII.

[0088] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein said Factor VII polypeptide ishuman Factor VIIa.

[0089] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein the ratio between the activity ofsaid Factor VII polypeptide and the activity of the native Factor VIIapolypeptide shown in SEQ ID NO:1 is at least about 1.25. In oneembodiment the ratio between the activity of said Factor VII polypeptideand the activity of the native Factor VIIa polypeptide shown in SEQ IDNO:1 is at least about 2.0. In a further embodiment the ratio betweenthe activity of said Factor VII polypeptide and the activity of thenative Factor VIIa polypeptide shown in SEQ ID NO:1 is at least about4.0.

[0090] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein the ratio between the activity ofsaid Factor VII polypeptide and the activity of the native Factor VIIapolypeptide shown in SEQ ID NO:1 is at least about 1.25 when tested in aFactor VIIa activity assay. In one embodiment the ratio between theactivity of said Factor VII polypeptide and the activity of the nativeFactor VIIa polypeptide shown in SEQ ID NO:1 is at least about 2.0 whentested in a Factor VIIa activity assay. In a further embodiment theratio between the activity of said Factor VII polypeptide and theactivity of the native Factor VIIa polypeptide shown in SEQ ID NO:1 isat least about 4.0 when tested in a Factor VIIa activity assay. TheFactor VIIa activity may be measured by the assays described in examples5 or 6.

[0091] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein the ratio between the activity ofsaid Factor VII polypeptide and the activity of the native Factor VIIapolypeptide shown in SEQ ID NO:1 is at least about 1.25 when tested inthe “In Vitro Hydrolysis Assay”. In one embodiment the ratio between theactivity of said Factor VII polypeptide and the activity of the nativeFactor VIIa polypeptide shown in SEQ ID NO:1 is at least about 2.0 whentested in the “In Vitro Hydrolysis Assay”. In a further embodiment theratio between the activity of said Factor VII polypeptide and theactivity of the native Factor VIIa polypeptide shown in SEQ ID NO:1 isat least about 4.0 when tested in the “In Vitro Hydrolysis Assay”.

[0092] In a further embodiment of the invention, the factor VIIpolypeptide is a polypeptide, wherein the ratio between the activity ofsaid Factor VII polypeptide and the activity of the native Factor VIIapolypeptide shown in SEQ ID NO:1 is at least about 1.25 when tested inthe “In Vitro Proteolysis Assay”. In one embodiment the ratio betweenthe activity of said Factor VII polypeptide and the activity of thenative Factor VIIa polypeptide shown in SEQ ID NO:1 is at least about2.0 when tested in the “In Vitro Proteolysis Assay”. In a furtherembodiment the ratio between the activity of said Factor VII polypeptideand the activity of the native Factor VIIa polypeptide shown in SEQ IDNO:1 is at least about 4.0 when tested in the “In Vitro ProteolysisAssay”. In a further embodiment the ratio between the activity of saidFactor VII polypeptide and the activity of the native Factor VIIapolypeptide shown in SEQ ID NO:1 is at least about 8.0 when tested inthe “In Vitro Proteolysis Assay”.

[0093] In a further embodiment of the invention, the factor VIIpolypeptide is human FVII with at least two substitutions relative tothe amino acid sequence of SEQ ID NO:1, wherein said substitutions are(i) L305V and (ii) one or more amino acids selected from the groupconsisting of K157X¹, K337A, D334X², S336X³, V158X⁴, E296V, and M298Q,wherein X¹ is Gly, Val, Ser, Thr, Asn, Gln, Asp, or Glu; X² is Gly orGlu; X³ is Gly or Glu; X⁴ is Thr or Asp.

[0094] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/K337A-FVII.

[0095] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158D-FVII.

[0096] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/E296V-FVII.

[0097] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/M298Q-FVII.

[0098] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158T-FVII.

[0099] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/K337A/V158T-FVII.

[0100] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/K337A/M298Q-FVII.

[0101] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/K337A/E296V-FVII.

[0102] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/K337A/V158D-FVII.

[0103] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158D/M2980-FVII.

[0104] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158D/E296V-FVII.

[0105] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158T/M298Q-FVII.

[0106] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158T/E296V-FVII.

[0107] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/E296V/M298Q-FVII.

[0108] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158D/E296V/M298Q-FVII.

[0109] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158T/E296V/M2980-FVII.

[0110] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158T/K337A/M2980-FVII.

[0111] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158T/E296V/K337A-FVII.

[0112] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158D/K337A/M298Q-FVII.

[0113] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/E296V/M298Q/K337A -FVII.

[0114] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158D/E296V/K337A -FVII.

[0115] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158D/E296V/M298Q/K337A-FVII.

[0116] In a further embodiment of the invention, the factor VIIpolypeptide is L305V/V158T/E296V/M298Q/K337A-FVII.

[0117] In a further aspect, the invention provides human coagulationFactor VIIa polypeptides that have increased tissue factor-independentactivity compared to native human coagulation Factor VIIa. In anotheraspect, the increased activity is not accompanied by changes in thesubstrate specificity. In another aspect of the invention, the bindingof the polypeptide variants to tissue factor should not be impaired andthe polypeptide variants should have at least the activity of wild-typeFactor VIIa when bound to tissue factor.

[0118] The terminology for amino acid substitutions used in thisdescription are as follows. The first letter represent the amino acidnaturally present at a position of SEQ ID NO:1. The following numberrepresent the position in SEQ ID NO:1. The second letter represent thedifferent amino acid substituting for the natural amino acid. An exampleis L305V/K337A-FVII, the leucine at position 305 of SEQ ID NO:1 isreplaced by a valine and the Lysine at position 337 of SEQ ID NO:1 isreplaced by an alanine, both mutations in the same Factor VIIpolypeptide variant.

[0119] In the present context the three-letter or one-letter indicationsof the amino acids have been used in their conventional meaning asindicated in table 1. Unless indicated explicitly, the amino acidsmentioned herein are L-amino acids. Further, the left and right ends ofan amino acid sequence of a peptide are, respectively, the N- andC-termini unless otherwise specified. TABLE 1 Abbreviations for aminoacids: Amino acid Tree-letter code One-letter code Glycine Gly G ProlinePro P Alanine Ala A Valine Val V Leucine Leu L Isoleucine Ile IMethionine Met M Cysteine Cys C Phenylalanine Phe F Tyrosine Tyr YTryptophan Trp W Histidine His H Lysine Lys K Arginine Arg R GlutamineGln Q Asparagine Asn N Glutamic Acid Glu E Aspartic Acid Asp D SerineSer S Threonine Thr T

[0120] Preparation of Factor VII Polypeptide Variants

[0121] The invention also relates to a method of preparing human FactorVII polypeptide variants as mentioned above. The Factor VII polypeptidevariants described herein may be produced by means of recombinantnucleic acid techniques. In general, a cloned wild-type Factor VIInucleic acid sequence is modified to encode the desired protein. Thismodified sequence is then inserted into an expression vector, which isin turn transformed or transfected into host cells. Higher eukaryoticcells, in particular cultured mammalian cells, are preferred as hostcells. The complete nucleotide and amino acid sequences for human FactorVII are known (see U.S. Pat. No. 4,784,950, where the cloning andexpression of recombinant human Factor VII is described). The bovineFactor VII sequence is described in Takeya et al., J. Biol. Chem.263:14868-14872 (1988)).

[0122] The amino acid sequence alterations may be accomplished by avariety of techniques. Modification of the nucleic acid sequence may beby site-specific mutagenesis. Techniques for site-specific mutagenesisare well known in the art and are described in, for example, Zoller andSmith (DNA 3:479-488, 1984) or “Splicing by extension overlap”, Hortonet al., Gene 77,1989, pp. 61-68. Thus, using the nucleotide and aminoacid sequences of Factor VII, one may introduce the alteration(s) ofchoice. Likewise, procedures for preparing a DNA construct usingpolymerase chain reaction using specific primers are well known topersons skilled in the art (cf. PCR Protocols, 1990, Academic Press, SanDiego, Calif., USA).

[0123] The nucleic acid construct encoding the Factor VII polypeptidevariant of the invention may suitably be of genomic or cDNA origin, forinstance obtained by preparing a genomic or cDNA library and screeningfor DNA sequences coding for all or part of the polypeptide byhybridization using synthetic oligonucleotide probes in accordance withstandard techniques (cf. Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd. Ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989).

[0124] The nucleic acid construct encoding the Factor VII polypeptidevariant may also be prepared synthetically by established standardmethods, e.g. the phosphoamidite method described by Beaucage andCaruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the methoddescribed by Matthes et al., EMBO Journal 3 (1984), 801-805. Accordingto the phosphoamidite method, oligonucleotides are synthesised, e.g. inan automatic DNA synthesiser, purified, annealed, ligated and cloned insuitable vectors. The DNA sequences encoding the human Factor VIIpolypeptide variants may also be prepared by polymerase chain reactionusing specific primers, for instance as described in U.S. Pat. No.4,683,202, Saiki et al., Science 239 (1988), 487-491, or Sambrook etal., supra.

[0125] Furthermore, the nucleic acid construct may be of mixed syntheticand genomic, mixed synthetic and cDNA or mixed genomic and cDNA originprepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate), the fragments corresponding to various parts of the entirenucleic acid construct, in accordance with standard techniques.

[0126] The nucleic acid construct is preferably a DNA construct. DNAsequences for use in producing Factor VII polypeptide variants accordingto the present invention will typically encode a pre-pro polypeptide atthe amino-terminus of Factor VII to obtain proper posttranslationalprocessing (e.g. gamma-carboxylation of glutamic acid residues) andsecretion from the host cell. The pre-pro polypeptide may be that ofFactor VII or another vitamin K-dependent plasma protein, such as FactorIX, Factor X, prothrombin, protein C or protein S. As will beappreciated by those skilled in the art, additional modifications can bemade in the amino acid sequence of the Factor VII polypeptide variantswhere those modifications do not significantly impair the ability of theprotein to act as a coagulant. For example, the Factor VII polypeptidevariants can also be modified in the activation cleavage site to inhibitthe conversion of zymogen Factor VII into its activated two-chain form,as generally described in U.S. Pat. No. 5,288,629, incorporated hereinby reference.

[0127] The DNA sequences encoding the human Factor VII polypeptidevariants are usually inserted into a recombinant vector which may be anyvector, which may conveniently be subjected to recombinant DNAprocedures, and the choice of vector will often depend on the host cellinto which it is to be introduced. Thus, the vector may be anautonomously replicating vector, i.e. a vector, which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated.

[0128] The vector is preferably an expression vector in which the DNAsequence encoding the human Factor VII polypeptide variants is operablylinked to additional segments required for transcription of the DNA. Ingeneral, the expression vector is derived from plasmid or viral DNA, ormay contain elements of both. The term, “operably linked” indicates thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in a promoter andproceeds through the DNA sequence coding for the polypeptide.

[0129] Expression vectors for use in expressing Factor VIIa polypeptidevariants will comprise a promoter capable of directing the transcriptionof a cloned gene or cDNA. The promoter may be any DNA sequence, whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell.

[0130] Examples of suitable promoters for directing the transcription ofthe DNA encoding the human Factor VI I polypeptide variant in mammaliancells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981),854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al.,Science 222 (1983), 809-814), the CMV promoter (Boshart et al., Cell41:521-530, 1985) or the adenovirus 2 major late promoter (Kaufman andSharp, Mol. Cell. Biol, 2:1304-1319, 1982).

[0131] An example of a suitable promoter for use in insect cells is thepolyhedrin promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al., FEBSLett. 311, (1992) 7-11), the P10 promoter (J. M. Vlak et al., J. Gen.Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosisvirus basic protein promoter (EP 397 485), the baculovirus immediateearly gene 1 promoter (U.S. Pat. Nos. 5,155,037; 5,162,222), or thebaculovirus 39K delayed-early gene promoter (U.S. Pat. Nos. 5,155,037;5,162,222).

[0132] Examples of suitable promoters for use in yeast host cellsinclude promoters from yeast glycolytic genes (Hitzeman et al., J. Biol.Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1(1982), 419-434) or alcohol dehydrogenase genes (Young et al., inGenetic Engineering of Microorganisms for Chemicals (Hollaender et al,eds.), Plenum Press, New York, 1982), or the TPI1 (U.S. Pat. No.4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652-654)promoters.

[0133] Examples of suitable promoters for use in filamentous fungus hostcells are, for instance, the ADH3 promoter (McKnight et al., The EMBO J.4 (1985), 2093-2099) or the tpiA promoter. Examples of other usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralα-amylase, A. niger acid stable α-amylase, A. niger or A. awamoriglucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkalineprotease, A. oryzae triose phosphate isomerase or A. nidulansacetamidase. Preferred are the TAKA-amylase and gluA promoters. Suitablepromoters are mentioned in, e.g. EP 238 023 and EP 383 779.

[0134] The DNA sequences encoding the human Factor VII polypeptidevariants may also, if necessary, be operably connected to a suitableterminator, such as the human growth hormone terminator (Palmiter etal., Science 222, 1983, pp. 809-814) or the TPI1 (Alber and Kawasaki, J.Mol. Appl. Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., The EMBOJ. 4, 1985, pp. 2093-2099) terminators. Expression vectors may alsocontain a set of RNA splice sites located downstream from the promoterand upstream from the insertion site for the Factor VII sequence itself.Preferred RNA splice sites may be obtained from adenovirus and/orimmunoglobulin genes. Also contained in the expression vectors is apolyadenylation signal located downstream of the insertion site.Particularly preferred polyadenylation signals include the early or latepolyadenylation signal from SV40 (Kaufman and Sharp, ibid.), thepolyadenylation signal from the adenovirus 5 Elb region, the humangrowth hormone gene terminator (DeNoto et al. Nucl. Acids Res.9:3719-3730, 1981) or the polyadenylation signal from the human FactorVII gene or the bovine Factor VII gene. The expression vectors may alsoinclude a noncoding viral leader sequence, such as the adenovirus 2tripartite leader, located between the promoter and the RNA splicesites; and enhancer sequences, such as the SV40 enhancer.

[0135] To direct the human Factor VII polypeptide variants of thepresent invention into the secretory pathway of the host cells, asecretory signal sequence (also known as a leader sequence, preprosequence or pre sequence) may be provided in the recombinant vector. Thesecretory signal sequence is joined to the DNA sequences encoding thehuman Factor VII polypeptide variants in the correct reading frame.Secretory signal sequences are commonly positioned 5′ to the DNAsequence encoding the peptide. The secretory signal sequence may bethat, normally associated with the protein or may be from a geneencoding another secreted protein.

[0136] For secretion from yeast cells, the secretory signal sequence mayencode any signal peptide, which ensures efficient direction of theexpressed human Factor VII polypeptide variants into the secretorypathway of the cell. The signal peptide may be naturally occurringsignal peptide, or a functional part thereof, or it may be a syntheticpeptide. Suitable signal peptides have been found to be the α-factorsignal peptide (cf. U.S. Pat. No. 4,870,008), the signal peptide ofmouse salivary amylase (cf. O. Hagenbuchle et al., Nature 289, 1981, pp.643-646), a modified carboxypeptidase signal peptide (cf. L. A. Valls etal., Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (cf. WO87/02670), or the yeast aspartic protease 3 (YAP3) signal peptide (cf.M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137).

[0137] For efficient secretion in yeast, a sequence encoding a leaderpeptide may also be inserted downstream of the signal sequence andupstream of the DNA sequence encoding the human Factor VII polypeptidevariants. The function of the leader peptide is to allow the expressedpeptide to be directed from the endoplasmic reticulum to the Golgiapparatus and further to a secretory vesicle for secretion into theculture medium (i.e. exportation of the human Factor VII polypeptidevariants across the cell wall or at least through the cellular membraneinto the periplasmic space of the yeast cell). The leader peptide may bethe yeast alpha-factor leader (the use of which is described in e.g.U.S. Pat. Nos. 4,546,082, 4,870,008, EP 16 201, EP 123 294, EP 123 544and EP 163 529). Alternatively, the leader peptide may be a syntheticleader peptide, which is to say a leader peptide not found in nature.Synthetic leader peptides may, for instance, be constructed as describedin WO 89/02463 or WO 92/11378.

[0138] For use in filamentous fungi, the signal peptide may convenientlybe derived from a gene encoding an Aspergillus sp. amylase orglucoamylase, a gene encoding a Rhizomucor miehei lipase or protease ora Humicola lanuginosa lipase. The signal peptide is preferably derivedfrom a gene encoding A. oryzae TAKA amylase, A. niger neutral α-amylase,A. niger acid-stable amylase, or A. niger glucoamylase. Suitable signalpeptides are disclosed in, e.g. EP 238 023 and EP 215 594.

[0139] For use in insect cells, the signal peptide may conveniently bederived from an insect gene (cf. WO 90/05783), such as the lepidopteranManduca sexta adipokinetic hormone precursor signal peptide (cf. U.S.Pat. No. 5,023,328).

[0140] The procedures used to ligate the DNA sequences coding for thehuman Factor VII polypeptide variants, the promoter and optionally theterminator and/or secretory signal sequence, respectively, and to insertthem into suitable vectors containing the information necessary forreplication, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y., 1989).

[0141] Methods of transfecting mammalian cells and expressing DNAsequences introduced in the cells are described in e.g. Kaufman andSharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol.Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci.USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro andPearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb,Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

[0142] Cloned DNA sequences are introduced into cultured mammalian cellsby, for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973) orelectroporation (Neumann et al., EMBO J. 1:841-845, 1982). To identifyand select cells that express the exogenous DNA, a gene that confers aselectable phenotype (a selectable marker) is generally introduced intocells along with the gene or cDNA of interest. Preferred selectablemarkers include genes that confer resistance to drugs such as neomycin,hygromycin, and methotrexate. The selectable marker may be anamplifiable selectable marker. A preferred amplifiable selectable markeris a dihydrofolate reductase (DHFR) sequence. Selectable markers arereviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers,Stoneham, Mass., incorporated herein by reference). The person skilledin the art will easily be able to choose suitable selectable markers.

[0143] Selectable markers may be introduced into the cell on a separateplasmid at the same time as the gene of interest, or they may beintroduced on the same plasmid. If, on the same plasmid, the selectablemarker and the gene of interest may be under the control of differentpromoters or the same promoter, the latter arrangement producing adicistronic message. Constructs of this type are known in the art (forexample, Levinson and Simonsen, U.S. Pat. No. 4,713,339). It may also beadvantageous to add additional DNA, known as “carrier DNA,” to themixture that is introduced into the cells.

[0144] After the cells have taken up the DNA, they are grown in anappropriate growth medium, typically 1-2 days, to begin expressing thegene of interest. As used herein the term “appropriate growth medium”means a medium containing nutrients and other components required forthe growth of cells and the expression of the human Factor VIIpolypeptide variants of interest. Media generally include a carbonsource, a nitrogen source, essential amino acids, essential sugars,vitamins, salts, phospholipids, protein and growth factors. Forproduction of gamma-carboxylated proteins, the medium will containvitamin K, preferably at a concentration of about 0.1 μg/ml to about 5μg/ml. Drug selection is then applied to select for the growth of cellsthat are expressing the selectable marker in a stable fashion. For cellsthat have been transfected with an amplifiable selectable marker thedrug concentration may be increased to select for an increased copynumber of the cloned sequences, thereby increasing expression levels.Clones of stably transfected cells are then screened for expression ofthe human Factor VII polypeptide variant of interest.

[0145] The host cell into which the DNA sequences encoding the humanFactor VII polypeptide variants is introduced may be any cell, which iscapable of producing the posttranslational modified human Factor VIIpolypeptide variants and includes yeast, fungi and higher eucaryoticcells.

[0146] Examples of mammalian cell lines for use in the present inventionare the COS-1 (ATCC CRL 1650), baby hamster kidney (BHK) and 293 (ATCCCRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. Apreferred BHK cell line is the tk⁻ ts13 BHK cell line (Waechter andBaserga, Proc. Natl. Acad. Sci. USA 79:1106-1110,1982, incorporatedherein by reference), hereinafter referred to as BHK 570 cells. The BHK570 cell line has been deposited with the American Type CultureCollection, 12301 Parklawn Dr., Rockville, Md. 20852, under ATCCaccession number CRL 10314. A tk⁻ ts13 BHK cell line is also availablefrom the ATCC under accession number CRL 1632. In addition, a number ofother cell lines may be used within the present invention, including RatHep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCCCCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin, Proc.Natl. Acad. Sci. USA 77:4216-4220, 1980).

[0147] Examples of suitable yeasts cells include cells of Saccharomycesspp. or Schizosaccharomyces spp., in particular strains of Saccharomycescerevisiae or Saccharomyces kluyveri. Methods for transforming yeastcells with heterologous DNA and producing heterologous polypeptidesthere from are described, e.g. in U.S. Pat. Nos. 4,599,311, 4,931,373,4,870,008, 5,037,743, and 4,845,075, all of which are herebyincorporated by reference. Transformed cells are selected by a phenotypedetermined by a selectable marker, commonly drug resistance or theability to grow in the absence of a particular nutrient, e.g. leucine. Apreferred vector for use in yeast is the POT1 vector disclosed in U.S.Pat. No. 4,931,373. The DNA sequences encoding the human Factor VIIpolypeptide variants may be preceded by a signal sequence and optionallya leader sequence, e.g. as described above. Further examples of suitableyeast cells are strains of Kluyveromyces, such as K. lactis, Hansenula,e.g. H. polymorpha, or Pichia, e.g. P. pastoris (cf. Gleeson et al., J.Gen. Microbiol. 132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279).

[0148] Examples of other fungal cells are cells of filamentous fungi,e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or Trichodermaspp., in particular strains of A. oryzae, A. nidulans or A. niger. Theuse of Aspergillus spp. for the expression of proteins is described in,e.g., EP 272 277, EP 238 023, EP 184 438 The transformation of F.oxysporum may, for instance, be carried out as described by Malardier etal., 1989, Gene 78: 147-156. The transformation of Trichoderma spp. maybe performed for instance as described in EP 244 234.

[0149] When a filamentous fungus is used as the host cell, it may betransformed with the DNA construct of the invention, conveniently byintegrating the DNA construct in the host chromosome to obtain arecombinant host cell. This integration is generally considered to be anadvantage as the DNA sequence is more likely to be stably maintained inthe cell. Integration of the DNA constructs into the host chromosome maybe performed according to conventional methods, e.g. by homologous orheterologous recombination.

[0150] Transformation of insect cells and production of heterologouspolypeptides therein may be performed as described in U.S. Pat. Nos.4,745,051; 4,879,236; 5,155,037; 5,162,222; EP 397,485) all of which areincorporated herein by reference. The insect cell line used as the hostmay suitably be a Lepidoptera cell line, such as Spodoptera frugiperdacells or Trichoplusia ni cells (cf. U.S. Pat. No. 5,077,214). Cultureconditions may suitably be as described in, for instance, WO 89/01029 orWO 89/01028, or any of the aforementioned references.

[0151] The transformed or transfected host cell described above is thencultured in a suitable nutrient medium under conditions permittingexpression of the human Factor VII polypeptide variant after which allor part of the resulting peptide may be recovered from the culture. Themedium used to culture the cells may be any conventional medium suitablefor growing the host cells, such as minimal or complex media containingappropriate supplements. Suitable media are available from commercialsuppliers or may be prepared according to published recipes (e.g. incatalogues of the American Type Culture Collection). The human FactorVII polypeptide variant produced by the cells may then be recovered fromthe culture medium by conventional procedures including separating thehost cells from the medium by centrifugation or filtration,precipitating the proteinaqueous components of the supernatant orfiltrate by means of a salt, e.g. ammonium sulphate, purification by avariety of chromatographic procedures, e.g. ion exchange chromatography,gelfiltration chromatography, affinity chromatography, or the like,dependent on the type of polypeptide in question.

[0152] Transgenic animal technology may be employed to produce theFactor VII polypeptide variants of the invention. It is preferred toproduce the proteins within the mammary glands of a host female mammal.Expression in the mammary gland and subsequent secretion of the proteinof interest into the milk overcomes many difficulties encountered inisolating proteins from other sources. Milk is readily collected,available in large quantities, and biochemically well characterized.Furthermore, the major milk proteins are present in milk at highconcentrations (typically from about 1 to 15 g/l).

[0153] From a commercial point of view, it is clearly preferable to useas the host a species that has a large milk yield. While smaller animalssuch as mice and rats can be used (and are preferred at the proof ofprinciple stage), it is preferred to use livestock mammals including,but not limited to, pigs, goats, sheep and cattle. Sheep areparticularly preferred due to such factors as the previous history oftransgenesis in this species, milk yield, cost and the readyavailability of equipment for collecting sheep milk (see, for example,WO 88/00239 for a comparison of factors influencing the choice of hostspecies). It is generally desirable to select a breed of host animalthat has been bred for dairy use, such as East Friesland sheep, or tointroduce dairy stock by breeding of the transgenic line at a laterdate. In any event, animals of known, good health status should be used.

[0154] To obtain expression in the mammary gland, a transcriptionpromoter from a milk protein gene is used. Milk protein genes includethose genes encoding caseins (see U.S. Pat. No. 5,304,489),beta-lactoglobulin, a-lactalbumin, and whey acidic protein. Thebeta-lactoglobulin (BLG) promoter is preferred. In the case of the ovinebeta-lactoglobulin gene, a region of at least the proximal 406 bp of 5′flanking sequence of the gene will generally be used, although largerportions of the 5′ flanking sequence, up to about 5 kbp, are preferred,such as a ˜4.25 kbp DNA segment encompassing the 5′ flanking promoterand non-coding portion of the beta-lactoglobulin gene (see Whitelaw etal., Biochem. J. 286: 31-39 (1992)). Similar fragments of promoter DNAfrom other species are also suitable.

[0155] Other regions of the beta-lactoglobulin gene may also beincorporated in constructs, as may genomic regions of the gene to beexpressed. It is generally accepted in the art that constructs lackingintrons, for example, express poorly in comparison with those thatcontain such DNA sequences (see Brinster et al., Proc. Natl. Acad. Sci.USA 85: 836-840 (1988); Palmiter et al., Proc. Natl. Acad. Sci. USA 88:478-482 (1991); Whitelaw et al., Transgenic Res. 1: 3-13 (1991); WO89/01343; and WO 91/02318, each of which is incorporated herein byreference). In this regard, it is generally preferred, where possible,to use genomic sequences containing all or some of the native introns ofa gene encoding the protein or polypeptide of interest, thus the furtherinclusion of at least some introns from, e.g, the beta-lactoglobulingene, is preferred. One such region is a DNA segment that provides forintron splicing and RNA polyadenylation from the 3′ non-coding region ofthe ovine beta-lactoglobulin gene. When substituted for the natural 3′non-coding sequences of a gene, this ovine beta-lactoglobulin segmentcan both enhance and stabilize expression levels of the protein orpolypeptide of interest. Within other embodiments, the regionsurrounding the initiation ATG of the variant Factor VII sequence isreplaced with corresponding sequences from a milk specific protein gene.Such replacement provides a putative tissue-specific initiationenvironment to enhance expression. It is convenient to replace theentire variant Factor VII pre-pro and 5′ non-coding sequences with thoseof, for example, the BLG gene, although smaller regions may be replaced.

[0156] For expression of Factor VII polypeptide variants in transgenicanimals, a DNA segment encoding variant Factor VII is operably linked toadditional DNA segments required for its expression to produceexpression units. Such additional segments include the above-mentionedpromoter, as well as sequences that provide for termination oftranscription and polyadenylation of mRNA. The expression units willfurther include a DNA segment encoding a secretory signal sequenceoperably linked to the segment encoding modified Factor VII. Thesecretory signal sequence may be a native Factor VII secretory signalsequence or may be that of another protein, such as a milk protein (see,for example, von Heijne, Nucl. Acids Res. 14: 4683-4690 (1986); andMeade et al., U.S. Pat. No. 4,873,316, which are incorporated herein byreference).

[0157] Construction of expression units for use in transgenic animals isconveniently carried out by inserting a variant Factor VII sequence intoa plasmid or phage vector containing the additional DNA segments,although the expression unit may be constructed by essentially anysequence of ligations. It is particularly convenient to provide a vectorcontaining a DNA segment encoding a milk protein and to replace thecoding sequence for the milk protein with that of a Factor VII variant;thereby creating a gene fusion that includes the expression controlsequences of the milk protein gene. In any event, cloning of theexpression units in plasmids or other vectors facilitates theamplification of the variant Factor VII sequence. Amplification isconveniently carried out in bacterial (e.g. E. coli) host cells, thusthe vectors will typically include an origin of replication and aselectable marker functional in bacterial host cells. The expressionunit is then introduced into fertilized eggs (including early-stageembryos) of the chosen host species. Introduction of heterologous DNAcan be accomplished by one of several routes, including microinjection(e.g. U.S. Pat. No. 4,873,191), retroviral infection (Jaenisch, Science240: 1468-1474 (1988)) or site-directed integration using embryonic stem(ES) cells (reviewed by Bradley et al., Bio/Technology 10: 534-539(1992)). The eggs are then implanted into the oviducts or uteri ofpseudopregnant females and allowed to develop to term. Offspringcarrying the introduced DNA in their germ line can pass the DNA on totheir progeny in the normal, Mendelian fashion, allowing the developmentof transgenic herds. General procedures for producing transgenic animalsare known in the art (see, for example, Hogan et al., Manipulating theMouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986;Simons et al., Bio/Technology 6: 179-183 (1988); Wall et al., Biol.Reprod. 32: 645-651 (1985); Buhler et al., Bio/Technology 8: 140-143(1990); Ebert et al., Bio/Technology 9: 835-838 (1991); Krimpenfort etal., Bio/Technology 9: 844-847 (1991); Wall et al., J. Cell. Biochem.49:113-120 (1992); U.S. Pat. Nos. 4,873,191; 4,873,316; WO 88/00239, WO90/05188, WO 92/11757; and GB 87/00458). Techniques for introducingforeign DNA sequences into mammals and their germ cells were originallydeveloped in the mouse (see, e.g., Gordon et al., Proc. Natl. Acad. Sci.USA 77: 7380-7384 (1980); Gordon and Ruddle, Science 214: 1244-1246(1981); Palmiter and Brinster, Cell 41: 343-345 (1985); Brinster et al.,Proc. Natl. Acad. Sci. USA 82: 4438-4442 (1985); and Hogan et al.(ibid.)). These techniques were subsequently adapted for use with largeranimals, including livestock species (see, e.g., WO 88/00239, WO90/05188, and WO 92/11757; and Simons et al., Bio/Technology 6: 179-183(1988)). To summarise, in the most efficient route used to date in thegeneration of transgenic mice or livestock, several hundred linearmolecules of the DNA of interest are injected into one of the pro-nucleiof a fertilized egg according to established techniques. Injection ofDNA into the cytoplasm of a zygote can also be employed.

[0158] Production in transgenic plants may also be employed. Expressionmay be generalised or directed to a particular organ, such as a tuber(see, Hiatt, Nature 344:469-479 (1990); Edelbaum et al., J. InterferonRes. 12:449-453 (1992); Sijmons et al., Bio/Technology 8:217-221 (1990);and EP 0 255 378).

[0159] The Factor VII polypeptide variants of the invention arerecovered from cell culture medium or milk. The Factor VII polypeptidevariants of the present invention may be purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J. -C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989). Preferably, they may be purified by affinity chromatographyon an anti-Factor VII antibody column. The use of calcium-dependentmonoclonal antibodies, as described by Wakabayashi et al., J. Biol.Chem. 261:11097-11108, (1986) and Thim et al., Biochemistry 27:7785-7793, (1988), is particularly preferred. Additional purificationmay be achieved by conventional chemical purification means, such ashigh performance liquid chromatography. Other methods of purification,including barium citrate precipitation, are known in the art, and may beapplied to the purification of the novel Factor VII polypeptide variantsdescribed herein (see, for example, Scopes, R., Protein Purification,Springer-Verlag, N.Y., 1982).

[0160] For therapeutic purposes it is preferred that the Factor VIIpolypeptide variants of the invention are substantially pure. Thus, in apreferred embodiment of the invention the Factor VII polypeptidevariants of the invention is purified to at least about 90 to 95%homogeneity, preferably to at least about 98% homogeneity. Purity may beassessed by e.g. gel electrophoresis and amino-terminal amino acidsequencing.

[0161] The Factor VII variant is cleaved at its activation site in orderto convert it to its two-chain form. Activation may be carried outaccording to procedures known in the art, such as those disclosed byOsterud, et al., Biochemistry 11:2853-2857 (1972); Thomas, U.S. Pat. No.4,456,591; Hedner and Kisiel, J. Clin. Invest. 71:1836-1841 (1983); orKisiel and Fujikawa, Behring Inst. Mitt. 73:29-42 (1983). Alternatively,as described by Bjoern et al. (Research Disclosure, 269 September 1986,pp. 564-565), Factor VII may be activated by passing it through anion-exchange chromatography column, such as Mono Q® (Pharmacia fineChemicals) or the like. The resulting activated Factor VII variant maythen be formulated and administered as described below.

[0162] Assays

[0163] The invention also provides suitable assays for selectingpreferred Factor VIIa variants according to the invention. These assayscan be performed as a simple preliminary in vitro test.

[0164] Thus, Example 5 herein discloses a simple test (entitled “inVitro Hydrolysis Assay”) for the activity of Factor VIIa variants of theinvention. Based thereon, Factor VIIa variants which are of particularinterest are such variants where the ratio between the activity of thevariant and the activity of native Factor VII shown in FIG. 1 is above1.0, e.g. at least about 1.25, preferably at least about 2.0, such as atleast about 3.0 or, even more preferred, at least about 4.0 when testedin the “In Vitro Hydrolysis Assay”.

[0165] The activity of the variants can also be measured using aphysiological substrate such as factor X (“in Vitro Proteolysis Assay”)(see Example 6), suitably at a concentration of 100-1000 nM, where thefactor Xa generated is measured after the addition of a suitablechromogenic substrate (eg. S-2765). In addition, the activity assay maybe run at physiological temperature.

[0166] The ability of the Factor VIIa variants to generate thrombin canalso be measured in an assay comprising all relevant coagulation factorsand inhibitors at physiological concentrations (minus factor VIII whenmimicking hemophilia A conditions) and activated platelets (as describedon p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547 whichis hereby incorporated as reference).

[0167] Administration and Pharmaceutical Compositions

[0168] The Factor VII polypeptide variants according to the presentinvention may be used to control bleeding disorders which have severalcauses such as clotting factor deficiencies (e.g. haemophilia A and B ordeficiency of coagulation factors XI or VII) or clotting factorinhibitors, or they may be used to control excessive bleeding occurringin subjects with a normally functioning blood clotting cascade (noclotting factor deficiencies or inhibitors against any of thecoagulation factors). The bleedings may be caused by a defectiveplatelet function, thrombocytopenia or von Willebrand's disease. Theymay also be seen in subjects in whom an increased fibrinolytic activityhas been induced by various stimuli.

[0169] In subjects who experience extensive tissue damage in associationwith surgery or vast trauma, the haemostatic mechanism may beoverwhelmed by the demand of immediate haemostasis and they may developbleedings in spite of a normal haemostatic mechanism. Achievingsatisfactory haemostasis is also a problem when bleedings occur inorgans such as the brain, inner ear region and eyes and may also be aproblem in cases of diffuse bleedings (haemorrhagic gastritis andprofuse uterine bleeding) when it is difficult to identify the source.The same problem may arise in the process of taking biopsies fromvarious organs (liver, lung, tumour tissue, gastrointestinal tract) aswell as in laparoscopic surgery. These situations share the difficultyof providing haemostasis by surgical techniques (sutures, clips, etc.).Acute and profuse bleedings may also occur in subjects on anticoagulanttherapy in whom a defective haemostasis has been induced by the therapygiven. Such subjects may need surgical interventions in case theanticoagulant effect has to be counteracted rapidly. Another situationthat may cause problems in the case of unsatisfactory haemostasis iswhen subjects with a normal haemostatic mechanism are givenanticoagulant therapy to prevent thromboembolic disease. Such therapymay include heparin, other forms of proteoglycans, warfarin or otherforms of vitamin K-antagonists as well as aspirin and other plateletaggregation inhibitors.

[0170] A systemic activation of the coagulation cascade may lead todisseminated intravascular coagulation (DIC). However, suchcomplications have not been seen in subjects treated with high doses ofrecombinant Factor VIIa because of a localised haemostatic process ofthe kind induced by the complex formation between Factor VIIa and TFexposed at the site of vessel wall injury. The Factor VII polypeptidevariants according to the invention may thus also be used in theiractivated form to control such excessive bleedings associated with anormal haemostatic mechanism.

[0171] For treatment in connection with deliberate interventions, theFactor VII polypeptide variants of the invention will typically beadministered within about 24 hours prior to performing the intervention,and for as much as 7 days or more thereafter. Administration as acoagulant can be by a variety of routes as described herein.

[0172] The dose of the Factor VII polypeptide variants ranges from about0.05 mg to 500 mg/day, preferably from about 1 mg to 200 mg/day, andmore preferably from about 10 mg to about 175 mg/day for a 70 kg subjectas loading and maintenance doses, depending on the weight of the subjectand the severity of the condition.

[0173] The pharmaceutical compositions are primarily intended forparenteral administration for prophylactic and/or therapeutic treatment.Preferably, the pharmaceutical compositions are administeredparenterally, i.e., intravenously, subcutaneously, or intramuscularly,or it may be administered by continuous or pulsatile infusion. Thecompositions for parenteral administration comprise the Factor VIIvariant of the invention in combination with, preferably dissolved in, apharmaceutically acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used, such as water, buffered water,0.4% saline, 0.3% glycine and the like. The Factor VII polypeptidevariants of the invention can also be formulated into liposomepreparations for delivery or targeting to the sites of injury. Liposomepreparations are generally described in, e.g., U.S. Pat. Nos. 4,837,028,4,501,728, and 4,975,282. The compositions may be sterilised byconventional, well-known sterilisation techniques. The resulting aqueoussolutions may be packaged for use or filtered under aseptic conditionsand lyophilised, the lyophilised preparation being combined with asterile aqueous solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc.

[0174] The concentration of Factor VII variant in these formulations canvary widely, i.e., from less than about 0.5% by weight, usually at or atleast about 1% by weight to as much as 15 or 20% by weight and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected.

[0175] Thus, a typical pharmaceutical composition for intravenousinfusion can be made up to contain 250 ml of sterile Ringer's solutionand 10 mg of the Factor VII variant. Actual methods for preparingparenterally administrable compositions will be known or apparent tothose skilled in the art and are described in more detail in, forexample, Remington's Pharmaceutical Sciences, 18th ed., Mack PublishingCompany, Easton, Pa. (1990).

[0176] The compositions containing the Factor VII polypeptide variantsof the present invention can be administered for prophylactic and/ortherapeutic treatments. In therapeutic applications, compositions areadministered to a subject already suffering from a disease, as describedabove, in an amount sufficient to cure, alleviate or partially arrestthe disease and its complications. An amount adequate to accomplish thisis defined as “therapeutically effective amount”. As will be understoodby the person skilled in the art amounts effective for this purpose willdepend on the severity of the disease or injury as well as the weightand general state of the subject. In general, however, the effectiveamount will range from about 0.05 mg up to about 500 mg of the FactorVII variant per day for a 70 kg subject, with dosages of from about 1.0mg to about 200 mg of the Factor VII variant per day being more commonlyused.

[0177] The FVIIa polypeptides of the present invention may generally beemployed in serious disease or injury states, that is, life threateningor potentially life threatening situations. In such cases, in view ofthe minimisation of extraneous substances and general lack ofimmunogenicity of human Factor VII polypeptide variants in humans, itmay be felt desirable by the treating physician to administer asubstantial excess of these variant Factor VII compositions.

[0178] In prophylactic applications, compositions containing the FactorVII variant of the invention are administered to a subject susceptibleto or otherwise at risk of a disease state or injury to enhance thesubject's own coagulative capability. Such an amount is defined to be a“prophylactically effective dose.” In prophylactic applications, theprecise amounts once again depend on the subject's state of health andweight, but the dose generally ranges from about 0.05 mg to about 500 mgper day for a 70-kilogram subject, more commonly from about 1.0 mg toabout 200 mg per day for a 70-kilogram subject.

[0179] Single or multiple administrations of the compositions can becarried out with dose levels and patterns being selected by the treatingphysician. For ambulatory subjects requiring daily maintenance levels,the Factor VII polypeptide variants may be administered by continuousinfusion using e.g. a portable pump system.

[0180] Local delivery of the Factor VII variant of the presentinvention, such as, for example, topical application may be carried out,for example, by means of a spray, perfusion, double balloon catheters,stent, incorporated into vascular grafts or stents, hydrogels used tocoat balloon catheters, or other well established methods. In any event,the pharmaceutical compositions should provide a quantity of Factor VIIvariant sufficient to effectively treat the subject.

[0181] The present invention is further illustrated by the followingexamples which, however, are not to be construed as limiting the scopeof protection. The features disclosed in the foregoing description andin the following examples may, both separately and in any combinationthereof, be material for realising the invention in diverse formsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0182]FIG. 1 shows the full amino acid sequence of native (wild type)human coagulation Factor VII (SEQ ID NO:1).

EXAMPLES

[0183] The terminology for amino acid substitutions used in thefollowing examples are as follows. The first letter represent the aminoacid naturally present at a position of SEQ ID NO:1. The followingnumber represent the position in SEQ ID NO:1. The second letterrepresent the different amino acid substituting for the natural aminoacid. An example is L305V/K337A-FVII, the leucine at position 305 of SEQID NO:1 is replaced by a valine and the Lysine at position 337 of SEQ IDNO:1 is replaced by an alanine, both mutations in the same Factor VIIvariant.

Example 1

[0184] DNA Encoding L305V/K337A-FVII, L305V/V158D/E296V/M298Q-FVII,L305V/V58D/E296V/M298Q/K337A-FVII.

[0185] DNA constructs encoding L305V/K337A-FVII,L305V/V158D/E296V/M298Q-FVII, and L305V/V158D/E296V/M298Q/K337A-FVIIwere prepared by site-directed mutagenesis using a supercoiled, doublestranded DNA vector with an insert of interest and two synthetic primerscontaining the desired mutation. The following primers were used: ForL305V-FVII: (SEQ ID NO:2) 5′-CGT GCC CCG GGT GAT GAC CCA GGA C-3′ (SEQID NO:3) 5′-GTC CTG GGT CAT CAC CCG GGG CAC G-3′ For K337A-FVII: (SEQ IDNO:4) 5′-CGG ATG GCA GCG CGG ACT CCT GCA AGG G-3′ (SEQ ID NO:5) 5′-CCCTTG CAG GAG TCC GCG CTG CCA TCC G-3′ For V158D-FVII: (SEQ ID NO:6)5′-GTG GGG GGC AAG GAC TGC CCC AAA GGG G-3′ (SEQ ID NO:7) 5′-CCC CTT TGGGGC AGT CCT TGC CCC CCA C-3′ For E296V/M298Q-FVII: (SEQ ID NO:8) 5′-GCCACG GCC CTG GTG CTC CAG GTC CTC AAC GTG CCC -3′ (SEQ ID NO:9) 5′-GGG CACGTT GAG GAC CTG GAG CAC CAG GGC CGT GGC -3′

[0186] The oligonucleotide primers, each complementary to oppositestrands of the vector, were extended during temperature cycling by meansof Pfu DNA polymerase. On incorporation of the primers, a mutatedplasmid containing staggered nicks was generated. Following temperaturecycling, the product was treated with DpnI which is specific formethylated and hemimethylated DNA to digest the parental DNA templateand to select for mutation-containing synthesized DNA.

[0187] Procedures for preparing a DNA construct using polymerase chainreaction using specific primers are well known to persons skilled in theart (cf. PCR Protocols, 1990, Academic Press, San Diego, Calif., USA).

Example 2

[0188] Preparation of L305V/K337A-FVII.

[0189] BHK cells were transfected essentially as previously described(Thim et al. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen(1996) FEBS Lett. 385, 241-243) to obtain expression of the variantL305V/K337A-FVII. The Factor VII variant was purified as follows:

[0190] Conditioned medium was loaded onto a 25-ml column of Q SepharoseFast Flow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% TritonX-100 and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water.

[0191] Elution of the protein was accomplished by stepping from 10 mMTris, 50 mM NaCl, 0.1% Triton X-100, pH 8.0 to 10 mM Tris, 50 mM NaCl,25 mM CaCl₂, 0.1% Triton X-100, pH 8.0. The fractions containingL305V/K337A-FVII were pooled and applied to a 25-ml column containingmonoclonal antibody F1A2 (Novo Nordisk, Bagsværd, Denmark) coupled toCNBr-activated Sepharose 4B (Pharmacia Biotech).

[0192] The column was equilibrated with 50 mM Hepes, pH 7.5, containing10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. After washing withequilibration buffer and equilibration buffer containing 2 M NaCl, boundmaterial was eluted with equilibration buffer containing 10 mM EDTAinstead of CaCl₂. Before use or storage, excess CaCl₂ over EDTA wasadded or L305V/K337A-FVII was transferred to a Ca²⁺-containing buffer.The yield of each step was followed by factor VII ELISA measurements andthe purified protein was analysed by SDS-PAGE.

Example 3

[0193] Preparation of L305V/V158D/E296V/M2980-FVII.

[0194] BHK cells were transfected essentially as previously described(Thim et al. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen(1996) FEBS Lett. 385, 241-243) to obtain expression of the variantL305V/V158D/E296V/M298Q-FVII. The Factor VII variant was purified asfollows:

[0195] Conditioned medium was loaded onto a 25-ml column of Q SepharoseFast Flow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% TritonX-100 and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by stepping from 10 mM Tris, 50 mM NaCl, 0.1% Triton X-100,pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaCl₂, 0.1% Triton X-100, pH8.0. The fractions containing L305V/V158D/E296V/M298Q-FVII were pooledand applied to a 25-ml column containing monoclonal antibody F1A2 (NovoNordisk, Bagsværd, Denmark) coupled to CNBr-activated Sepharose 4B(Pharmacia Biotech).

[0196] The column was equilibrated with 50 mM Hepes, pH 7.5, containing10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. After washing withequilibration buffer and equilibration buffer containing 2 M NaCl, boundmaterial was eluted with equilibration buffer containing 10 mM EDTAinstead of CaCl₂. Before use or storage, excess CaCl₂ over EDTA wasadded or L305V/V158D/E296V/M2980-FVII was transferred to aCa²⁺-containing buffer. The yield of each step was followed by factorVII ELISA measurements and the purified protein was analysed bySDS-PAGE.

Example 4

[0197] Preparation of L305V/V158D/E296V/M298Q/K337A-FVII.

[0198] BHK cells were transfected essentially as previously described(Thim et al. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen(1996) FEBS Lett. 385, 241-243) to obtain expression of the variantL305V/V158D/E296V/M2980/K337A-FVII. The Factor VII variant was purifiedas follows:

[0199] Conditioned medium was loaded onto a 25-ml column of Q SepharoseFast Flow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% TritonX-100 and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by stepping from 10 mM Tris, 50 mM NaCl, 0.1% Triton X-100,pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaCl₂, 0.1% Triton X-100, pH8.0. The fractions containing L305V/V158D/E296V/M2980/K337A-FVII werepooled and applied to a 25-ml column containing monoclonal antibody F1A2(Novo Nordisk, Bagsværd, Denmark) coupled to CNBr-activated Sepharose 4B(Pharmacia Biotech).

[0200] The column was equilibrated with 50 mM Hepes, pH 7.5, containing10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. After washing withequilibration buffer and equilibration buffer containing 2 M NaCl, boundmaterial was eluted with equilibration buffer containing 10 mM EDTAinstead of CaCl₂. Before use or storage, excess CaCl₂ over EDTA wasadded or L305V/V158D/E296V/M2980/K337A-FVII was transferred to aCa²⁺-containing buffer. The yield of each step was followed by factorVII ELISA measurements and the purified protein was analysed bySDS-PAGE.

Example 5

[0201] In vitro Hydrolysis Assay

[0202] Native (wild-type) Factor VIIa and Factor VIIa variant (bothhereafter referred to as “Factor VIIa”) are assayed in parallel todirectly compare their specific activities. The assay is carried out ina microtiter plate (MaxiSorp, Nunc, Denmark). The chromogenic substrateD-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), finalconcentration 1 mM, is added to Factor VIIa (final concentration 100 nM)in 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 5 mM CaCl₂ and 1 mg/mlbovine serum albumin. The absorbance at 405 nm is measured continuouslyin a SpectraMax™ 340 plate reader (Molecular Devices, USA). Theabsorbance developed during a 20-minute incubation, after subtraction ofthe absorbance in a blank well containing no enzyme, is used tocalculate the ratio between the activities of variant and wild-typeFactor VIIa:

Ratio=(A _(405 nm) Factor VIIa variant)/(A _(405 nm) Factor VIIawild-type).

Example 6

[0203] In vitro Proteolysis Assay

[0204] Native (wild-type) Factor VIIa and Factor VIIa variant (bothhereafter referred to as “Factor VIIa”) are assayed in parallel todirectly compare their specific activities. The assay is carried out ina microtiter plate (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nM) andFactor X (0.8 microM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1M NaCl, 5 mM CaCl₂ and 1 mg/ml bovine serum albumin, are incubated for15 min. Factor X cleavage is then stopped by the addition of 50 microL50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 20 mM EDTA and 1 mg/mlbovine serum albumin. The amount of Factor Xa generated is measured byaddition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide(S-2765, Chromogenix, Sweden), final concentration 0.5 mM. Theabsorbance at 405 nm is measured continuously in a SpectraMax™ 340 platereader (Molecular Devices, USA). The absorbance developed during 10minutes, after subtraction of the absorbance in a blank well containingno FVIIa, is used to calculate the ratio between the proteolyticactivities of variant and wild-type Factor VIIa:

Ratio=(A _(405 nm) Factor VIIa variant)/(A _(405 nm) Factor VIIawild-type). EXAMPLE 7 RELATIVE ACTIVITIES OF FVIIA VARIANTS MEASURED INTHE ASSAYS DESCRIBED IN EXAMPLES 5 AND 6. Ratio in Ratio in Variantexample 5 example 6 L305V/K337A-FVII 7.2 6.2L305V/V158D/E296V/M298Q-FVII 6.7 45 L305V/V158D/E296V/M298Q/K337A-FVII11.5 72 wt-FVIIa 1.0 1.0

[0205]

0 SEQUENCE LISTING (The amino acid sequence of native human coagulationFactor VII):Ala-Asn-Ala-Phe-Leu-GLA-GLA-Leu-Arg-Pro-G1y-Ser-Leu-GLA-Arg-GLA-Cys-Lys-SEQ ID NO. 1 5 10 15GLA-GLA-Gln-Cys-Ser-Phe-GLA-GLA-Ala-Arg-GLA-Ile-Phe-Lys-Asp-Ala-GLA-Arg-20 25 30 35Thr-Lys-Leu-Phe-Trp-Ile-Ser-Tyr-Ser-Asp-Gly-Asp-Gln-Cys-Ala-Ser-Ser-Pro-40 45 50Cys-Gln-Asn-Gly-Gly-Ser-Cys-Lys-Asp-Gln-Leu-Gln-Ser-Tyr-Ile-Cys-Phe-Cys-55 60 65 70Leu-Pro-Ala-Phe-Glu-Gly-Arg-Asn-Cys-Glu-Thr-His-Lys-Asp-Asp-Gln-Leu-Ile-75 80 85 90Cys-Val-Asn-Glu-Asn-Gly-Gly-Cys-Glu-Gln-Tyr-Cys-Ser-Asp-His-Thr-Gly-Thr-95 100 105Lys-Arg-Ser-Cys-Arg-Cys-His-Glu-Gly-Tyr-Ser-Leu-Leu-Ala-Asp-Gly-Val-Ser-110 115 120 125Cys-Thr-Pro-Thr-Val-Glu-Tyr-Pro-Cys-Gly-Lys-Ile-Pro-Ile-Leu-Glu-Lys-Arg-130 135 140Asn-Ala-Ser-Lys-Pro-Gln-Gly-Arg-Ile-Val-Gly-Gly-Lys-Val-Cys-Pro-Lys-Gly-145 150 155 160Glu-Cys-Pro-Trp-Gln-Val-Leu-Leu-Leu-Val-Asn-Gly-Ala-Gln-Leu-Cys-Gly-Gly-165 170 175Thr-Leu-Ile-Asn-Thr-Ile-Trp-Val-Val-Ser-Ala-Ala-His-Cys-Phe-Asp-Lys-Ile-185 190 195Lys-Asn-Trp-Arg-Asn-Leu-Ile-Ala-Val-Leu-Gly-Glu-His-Asp-Leu-Ser-Glu-His-200 205 210Asp-Gly-Asp-Glu-Gln-Ser-Arg-Arg-Val-Ala-Gln-Val-Ile-Ile-Pro-Ser-Thr-Tyr-220 225 230Val-Pro-Gly-Thr-Thr-Asn-His-Asp-Ile-Ala-Leu-Leu-Arg-Leu-His-Gln-Pro-Val-235 240 245 250Val-Leu-Thr-Asp-His-Val-Val-Pro-Leu-Cys-Leu-Pro-Glu-Arg-Thr-Phe-Ser-Glu-255 260 265 270Arg-Thr-Leu-Ala-Phe-Val-Arg-Phe-Ser-Leu-Val-Ser-Gly-Trp-Gly-Gln-Leu-Leu-275 280 285Asp-Arg-Gly-Ala-Thr-Ala-Leu-Glu-Leu-Met-Val-Leu-Asn-Val-Pro-Arg-Leu-Met-290 295 300 305 306Thr-Gln-Asp-Cys-Leu-Gln-Gln-Ser-Arg-Lys-Val-Gly-Asp-Ser-Pro-Asn-Ile-Thr-310 315 320Glu-Tyr-Met-Phe-Cys-Ala-Gly-Tyr-Ser-Asp-Gly-Ser-Lys-Asp-Ser-Cys-Lys-Gly-325 330 335 340Asp-Ser-Gly-Gly-Pro-His-Ala-Thr-His-Tyr-Arg-Gly-Thr-Trp-Tyr-Leu-Thr-Gly-345 350 355 360Ile-Val-Ser-Trp-Gly-Gln-Gly-Cys-Ala-Thr-Val-Gly-His-Phe-Gly-Val-Tyr-Thr-365 370 375Arg-Val-Ser-Gln-Tyr-Ile-Glu-Trp-Leu-Gln-Lys-Leu-Met-Arg-Ser-Glu-Pro-Arg-380 385 390 395 Pro-Gly-Val-Leu-Leu-Arg-Ala-Pro-Phe-Pro 400 405 406 (DNAprimer for preparation of L305V-FVII): 5′-CGT GCC CCG GGT GAT GAC CCAGGA C-3′ SEQ ID NO:2 (DNA primer for preparation of L305V-FVII): 5′-GTCCTG GGT CAT CAC CCG GGG CAC G-3′ SEQ ID NO:3 (DNA primer for preparationof K337A-FVII): 5′-CGG ATG GCA GCG CGG ACT CCT GCA AGG G-3′ SEQ ID NO:4(DNA primer for preparation of K337A-FVII): 5′-CCC TTG CAG GAG TCC GCGCTG CCA TCC G-3′ SEQ ID NO:5 (DNA primer for preparation of V158D-FVII):5′-GTG GGG GGC AAG GAC TGC CCC AAA GGG G-3′ SEQ ID NO:6 (DNA primer forpreparation of V158D-FVII): 5′-CCC CTT TGG GGC AGT CCT TGC CCC CCA C-3′SEQ ID NO:7 (DNA primer for preparation of E296V/M298Q-FVII): 5′-GCC ACGGCC CTG GTG CTC CAG GTC CTC AAC GTG CCC-3′ SEQ ID NO:8 (DNA primer forpreparation of E296V/M298Q-FVII): 5′-GGG CAC GTT GAG GAC CTG GAG CAC CAGGGC CGT GGC-3′ SEQ ID NQ:9

1. A Factor VII polypeptide comprising at least two substitutionsrelative to the amino acid sequence of SEQ ID NO:1, wherein saidsubstitutions are (i) replacement of L305 with any other amino acid, and(ii) replacement with any other amino acid of one or more amino acidsselected from the group consisting of K157, K337, D334, S336, V158,E296, and M298.
 2. The Factor VII polypeptide according to claim 1,wherein L305 is replaced with any other amino acid and K157 is replacedwith any other amino acid.
 3. The Factor VII polypeptide according toclaim 1, wherein L305 is replaced with any other amino acid and K337 isreplaced with any other amino acid.
 4. The Factor VII polypeptideaccording to claim 1, wherein L305 is replaced with any other amino acidand D334 is replaced with any other amino acid.
 5. The Factor VIIpolypeptide according to claim 1, wherein L305 is replaced with anyother amino acid and S336 is replaced with any other amino acid.
 6. TheFactor VII polypeptide according to claim 1, wherein L305 is replacedwith any other amino acid and V158 is replaced with any other aminoacid.
 7. The Factor VII polypeptide according to claim 1, wherein L305is replaced with any other amino acid and E296 is replaced with anyother amino acid.
 8. The Factor VII polypeptide according to claim 1,wherein L305 is replaced with any other amino acid and M298 is replacedwith any other amino acid.
 9. The Factor VII polypeptide according toclaim 1, wherein at least one amino acid in the remaining positions inthe protease domain has been replaced with any other amino acid.
 10. TheFactor VII polypeptide according to claim 9, wherein at the most 20additional amino acids in the remaining positions in the protease domainhave been replaced with any other amino acids.
 11. The Factor VIIpolypeptide according to claim 9, wherein at least one amino acidcorresponding to an amino acid at a position selected from 159-170 ofSEQ ID NO:1 has been replaced with any other amino acid.
 12. The FactorVII polypeptide according to claim 9, wherein at least one amino acidcorresponding to an amino acid at a position selected from 290-304 ofSEQ ID NO:1 has been replaced with any other amino acid.
 13. The FactorVII polypeptide according to claim 12, wherein R304 has been replaced byan amino acid selected from the group consisting of Tyr, Phe, Leu, andMet.
 14. The Factor VII polypeptide according to claim 9, wherein atleast one amino acid corresponding to an amino acid at a positionselected from 306-312 of SEQ ID NO:1 has been replaced with any otheramino acid.
 15. The Factor VII polypeptide according to claim 14,wherein M306 has been replaced by an amino acid selected from the groupconsisting of Asp, and Asn.
 16. The Factor VII polypeptide according toclaim 14, wherein D309 has been replaced by an amino acid selected fromthe group consisting of Ser and Thr.
 17. The Factor VII polypeptideaccording to claim 9, wherein at least one amino acid corresponding toan amino acid at a position selected from 330-339 of SEQ ID NO:1 hasbeen replaced with any other amino acid.
 18. The Factor VII polypeptideaccording to claim 9, wherein A274 has been replaced with any otheramino acid.
 19. The Factor VII polypeptide according to claim 18,wherein said A274 has been replaced by an amino acid selected from thegroup consisting of Met, Leu, Lys, and Arg.
 20. The Factor VIIpolypeptide according to claim 1, wherein said polypeptide contains twosubstitutions relative to the amino acid sequence of SEQ ID NO:1 andwherein said substitutions are (i) replacement of L305 with any otheramino acid and (ii) replacement with any other amino acid of one aminoacid selected from the group consisting of K157, K337, D334, S336, V158,E296, and M298.
 21. The Factor VII polypeptide according to claim 1,wherein said polypeptide contains three substitutions relative to theamino acid sequence of SEQ ID NO:1 and wherein said substitutions are(i) replacement of L305 with any other amino acid and (ii) replacementwith any other amino acid of two amino acids selected from the groupconsisting of K157, K337, D334, S336, V158, E296, and M298.
 22. TheFactor VII polypeptide according to claim 1, wherein said polypeptidecontains four substitutions relative to the amino acid sequence of SEQID NO:1 and wherein said substitutions are (i) replacement of L305 withany other amino acid and (ii) replacement with any other amino acid ofthree amino acids selected from the group consisting of K157, K337,D334, S336, V158, E296, and M298.
 23. The Factor VII polypeptideaccording to claim 1, wherein said polypeptide contains fivesubstitutions relative to the amino acid sequence of SEQ ID NO:1 andwherein said substitutions are (i) replacement of L305 with any otheramino acid and (ii) replacement with any other amino acid of four aminoacids selected from the group consisting of K157, K337, D334, S336,V158, E296, and M298.
 24. The Factor VII polypeptide according to claim1, wherein said polypeptide contains six substitutions relative to theamino acid sequence of SEQ ID NO:1 and wherein said substitutions are(i) replacement of L305 with any other amino acid and (ii) replacementwith any other amino acid of five amino acids selected from the groupconsisting of K157, K337, D334, S336, V158, E296, and M298.
 25. TheFactor VII polypeptide according to claim 1, wherein said polypeptidecontains seven substitutions relative to the amino acid sequence of SEQID NO:1 and wherein said substitutions are (i) replacement of L305 withany other amino acid and (ii) replacement with any other amino acid ofsix amino acids selected from the group consisting of K157, K337, D334,S336, V158, E296, and M298.
 26. The Factor VII polypeptide according toclaim 1, wherein said polypeptide contains eight substitutions relativeto the amino acid sequence of SEQ ID NO:1 and wherein said substitutionsare (i) replacement of L305 with any other amino acid and (ii)replacement with any other amino acid of the amino acids K157, K337,D334, S336, V158, E296, and M298.
 27. The Factor VII polypeptideaccording to claim 1, wherein said K157 has been replaced by an aminoacid selected from the group consisting of Gly, Val, Ser, Thr, Asn, Gln,Asp, and Glu.
 28. The Factor VII polypeptide according to claim 1,wherein said K337 has been replaced by an amino acid selected from thegroup consisting of Ala, Gly, Val, Ser, Thr, Asn, Gln, Asp, and Glu. 29.The Factor VII polypeptide according to claim 1, wherein said D334 hasbeen replaced by an amino acid selected from the group consisting ofGly, and Glu.
 30. The Factor VII polypeptide according to claim 1,wherein said S336 has been replaced by an amino acid selected from thegroup consisting of Gly, and Glu.
 31. The Factor VII polypeptideaccording to claim 1, wherein said V158 has been replaced by an aminoacid selected from the group consisting of Ser, Thr, Asn, Gln, Asp, andGlu.
 32. The Factor VII polypeptide according to claim 1, wherein saidE296 has been replaced by an amino acid selected from the groupconsisting of Arg, Lys, and Val.
 33. The Factor VII polypeptideaccording to claim 1, wherein said M298 has been replaced by an aminoacid selected from the group consisting of Lys, Arg, Gln, and Asn. 34.The Factor VII polypeptide according to claim 1, wherein said L305 hasbeen replaced by an amino acid selected from the group consisting ofVal, Tyr and Ile.
 35. The Factor VII polypeptide according to claim 34,wherein said L305 has been replaced by Val.
 36. The Factor VIIpolypeptide according to claim 1, wherein the amino acid has beenreplaced with any other amino acid which can be encoded bypolynucleotide constructs.
 37. The Factor VII polypeptide according toclaim 1, wherein said Factor VII polypeptide is human Factor VII. 38.The Factor VII polypeptide according to claim 1, wherein said Factor VIIpolypeptide is human Factor VIIa.
 39. The Factor VII polypeptideaccording to claim 1, wherein the ratio between the activity of saidFactor VII polypeptide and the activity of the native Factor VIIapolypeptide shown in SEQ ID NO:1 is at least about 1.25.
 40. The FactorVII polypeptide according to claim 39, wherein said ratio is at leastabout 2.0.
 41. The Factor VII polypeptide according to claim 20, whichis L305V/K337A-FVII.
 42. The Factor VII polypeptide according to claim22, which is L305V/V158D/E296V/M298Q-FVII.
 43. The Factor VIIpolypeptide according to claim 23, which isL305V/V158D/E296V/M298Q/K337A-FVII.
 44. A polynucleotide constructencoding a Factor VII polypeptide according to claim
 1. 45. Thepolynucleotide construct according to claim 44, wherein said constructis a vector.
 46. A host cell comprising the polynucleotide constructaccording to claim
 44. 47. The host cell according to claim 46, which isa eukaryotic cell.
 48. The host cell according to claim 47, which is ofmammalian origin.
 49. The host cell according to claim 48, wherein thecell is selected from the group consisting of CHO cells, HEK cells andBHK cells.
 50. A transgenic animal containing and expressing thepolynucleotide construct as defined in claim
 44. 51. A transgenic plantcontaining and expressing the polynucleotide construct as defined inclaim
 44. 52. A method for producing the Factor VII polypeptide, themethod comprising (i) cultivating a cell as defined in claim 46 in anappropriate growth medium under conditions allowing expression of thepolynucleotide construct and (ii) recovering the resulting polypeptidefrom the culture medium.
 53. A method for producing the Factor VIIpolypeptide, the method comprising recovering the Factor VII polypeptidefrom milk produced by the transgenic animal defined in claim
 50. 54. Amethod for producing the Factor VII polypeptide, the method comprising(i) cultivating a cell of a transgenic plant as defined in claim 51, and(ii) recovering the Factor VII polypeptide from the plant.
 55. Apharmaceutical composition comprising a Factor VII polypeptidecomprising at least two substitutions relative to the amino acidsequence of SEQ ID NO:1, wherein said substitutions are (i) replacementof L305 with any other amino acid, and (ii) replacement with any otheramino acid of one or more amino acids selected from the group consistingof K157, K337, D334, S336, V158, E296, and M298; and, optionally, apharmaceutically acceptable carrier.
 56. A pharmaceutical compositioncomprising a Factor VII polypeptide as defined in any of claims 1-43,and, optionally, a pharmaceutically acceptable carrier.
 57. Use of aFactor VII polypeptide comprising at least two substitutions relative tothe amino acid sequence of SEQ ID NO:1, wherein said substitutions are(i) replacement of L305 with any other amino acid, and (ii) replacementwith any other amino acid of one or more amino acids selected from thegroup consisting of K157, K337, D334, S336, V158, E296, and M298; forthe preparation of a medicament.
 58. Use of a Factor VII polypeptideaccording to claim 57, wherein the medicament is for the treatment ofbleeding disorders or bleeding episodes or for the enhancement of thenormal haemostatic system.
 59. Use of a Factor VII polypeptide asdefined in any of claims 1-43 for the preparation of a medicament forthe treatment of bleeding disorders or bleeding episodes or for theenhancement of the normal haemostatic system.
 60. Use according to anyof claims 57-59 for the treatment of haemophilia A or B.
 61. A methodfor the treatment of bleeding disorders or bleeding episodes in asubject or for the enhancement of the normal haemostatic system, themethod comprising administering a therapeutically or prophylacticallyeffective amount of a Factor VII polypeptide comprising at least twosubstitutions relative to the amino acid sequence of SEQ ID NO:1,wherein said substitutions are (i) replacement of L305 with any otheramino acid, and (ii) replacement with any other amino acid of one ormore amino acids selected from the group consisting of K157, K337, D334,S336, V158, E296, and M298; to a subject in need thereof.
 62. A methodfor the treatment of bleeding disorders or bleeding episodes in asubject or for the enhancement of the normal haemostatic system, themethod comprising administering a therapeutically or prophylacticallyeffective amount of a Factor VII polypeptide as defined in any of claims1-43 to a subject in need thereof.
 63. A Factor VII polypeptide asdefined in any of claims 1-43 for use as a medicament.